The long term objective of my research is to determine the mechanisms that control the initiation of protein synthesis in yeast. The goal of this proposal is to describe in genetic and molecular terms components of the translation initiation complex that function in mediating ribosomal binding of the 5' end of mRNA, scanning of the leader and recognition of the initiator region and the mechanisms by which these components may control the initiation process. The following specific aims are directed at achieving this goal: 1) External suppressors will be isolated for their ability to overcome a block in translation initiation at HIS4 as a result of hairpin loop structures in the 5' non-coding region of the mRNA. These suppressors in conjunction with the previously isolates susl and sus2 suppressor loci (suppressor of secondary structure) will be characterized at the molecular and biochemical level to determine if these gene products function at the time of ribosomal binding or scanning of mRNA. 2) The previously isolated suil external suppressor of initiator codon mutations at HIS4 will be characterized at the molecular and biochemical level to determine its role in start site selection as was established for the sui2 and SUI3 suppressors loci. Furthermore, we will initiate the screening and characterization of recessive lethal suppressors of initiator codon mutants obtained by reversion analysis of diploid yeast strains to determine if other components of the initiation complex might mediate ribosomal recognition of the initiator region during the scanning process. 3) The functional significance of a zinc II finger motif in the SUI3 suppressor gene which encodes the Beta-subunit of the eukaryotic initiation factor 2, eIF-2, will be addressed by genetic and molecular approaches. In addition, we will employ direct genetic and molecular approaches to determine if the phosphorylation of the alpha-subunit of eIF-2 functions in controlling the initiation of protein synthesis in yeast as believed for the mammalian eIF-2 alpha subunit that shows considerable homology. Based on similarities between the yeast and mammalian initiation process and homologies between yeast and human initiation factors that we have established, these studies should be relevant to the basic mechanisms that mediate the scanning process in all eukaryotes and that function in coordination normal cellular growth.